The Rpb9 Subunit of RNA Polymerase II Binds Transcription Factor TFIIE and Interferes with the SAGA and Elongator Histone Acetyltransferases

Abstract

Rpb9 is a small subunit of yeast RNA polymerase II participating in elongation and formed of two conserved zinc domains. rpb9 mutants are viable, with a strong sensitivity to nucleotide-depleting drugs. Deleting the C-terminal domain down to the first 57 amino acids has no detectable growth defect. Thus, the critical part of Rpb9 is limited to a N-terminal half that contacts the lobe of the second largest subunit (Rpb2) and forms a ␤-addition motif with the "jaw" of the largest subunit (Rpb1). Rpb9 has homology to the TFIIS elongation factor, but mutants inactivated for both proteins are indistinguishable from rpb9 single mutants. In contrast, rpb9 mutants are lethal in cells lacking the histone acetyltransferase activity of the RNA polymerase II Elongator and SAGA factors. In a two-hybrid test, Rpb9 physically interacts with Tfa1, the largest subunit of TFIIE. The interacting fragment, comprising amino acids 62-164 of Tfa1, belongs to a conserved zinc motif. Tfa1 is immunoprecipitated by RNA polymerase II. This co-purification is strongly reduced in rpb9-⌬, suggesting that Rpb9 contributes to the recruitment of TFIIE on RNA polymerase II.The recent determination of the bacterial RNA polymerase (1) and yeast RNA polymerase II (Pol II) 1 (2) core structures has opened a new chapter in transcription studies. The remarkably similar organization of these two enzymes leaves no doubt as to the strong mechanistic conservation of the transcription process. This similarity was anticipated from the sequence homology existing between the ␤Ј␤␣ 2 bacterial core enzyme and 5 of the 10 core subunits of Pol II. Rpb1 and Rpb2 are homologues of ␤Ј and ␤, the Rpb3/Rpb11 heterodimer corresponding to the bacterial ␣2 dimer, and is distantly related to Rpb6 (3). On the other hand, Pol II contains five small subunits (Rpb5, Rpb8, Rpb9, Rpb10, Rpb12) not found in the bacterial enzyme. These subunits also belong to the core structure of Pol I and Pol III or are related to it, in the case of Rpb9. Except for Rpb8, they are also akin to archaeal polypeptides (4, 5).The present study deals with the Rpb9 subunit, belonging to a conserved family of eukaryotic and archeal zinc-binding polypeptides that also includes the yeast Pol I (Rpa12 (6)) and Pol III (Rpc11 (7)) subunits and the TFIIS elongation factor (8 -10) encoded by PPR2 in Saccharomyces cerevisiae (11,12). There is ample evidence that Rpb9 (9), Rpc11 (7), and TFIIS (9, 13) control transcription elongation by activating the RNA cleavage activity inherent to all RNA polymerases. The fact that yeast ppr2 (14), rpb9 (15), and rpa12 (16) mutants are strongly sensitive to nucleotide-depleting drugs is also consistent with a major elongation defect. Rpb9 is highly conserved in evolution, and the yeast subunit can be replaced in vivo by its human counterpart (17). However, animal mutants (Drosophila melanogaster) are lethal (18), whereas yeast null mutants have only a limited growth defect (19).Rpa12, Rpb9, and Rpc11 have a related organization in Pol I, Pol II, and Pol III, respectively. ...

TFIID and Spt-Ada-Gcn5-Acetyltransferase Functions Probed by Genome-wide Synthetic Genetic Array Analysis Using a Saccharomyces cerevisiae taf9-ts Allele

“…This raises the possibility that TAF9-interacting genes for regulators of transcription elongation mostly represent SAGA-specific interactions, while those for Mediator and most chromatin modification/remodeling complexes largely represent TFIID-specific interactions. Supporting this notion, several studies are consistent with a role of SAGA in elongation (Van Mullem et al 2002;Rodriguez-Navarro et al 2004;Wery et al 2004), including the fact that SAGA physically and functionally interacts with both the transcription elongation factor TFIIS (Wery et al 2004) and the PolII subunit Rpb9, which has been linked to elongation control (Van Mullem et al 2002). Hence, to further examine possible involvement of SAGA in transcriptional elongation, we used ChIP to analyze the integrity of nucleosomes within the coding sequence of a gene known to be controlled by SAGA, PDR5 (Gao et al 2004).…”

Yeast Rpb9 Plays an Important Role in Ubiquitylation and Degradation of Rpb1 in Response to UV-Induced DNA Damage

“…In addition to its TCR function, Rpb9 has multiple transcription-related functions, such as selection of correct transcription start site (8,15,58), transcription elongation (12,49), and maintenance of transcription fidelity (29). In this paper, we present evidence that Rpb9 also plays an important role in Rpb1 ubiquitylation and degradation in response to UV radiation.…”

“…5A) (5). The Zn1 and linker domains are required for transcription elongation (22,49) and TCR functions (22), whereas the entire Zn2 domain is dispensable for these functions in vivo. However, both Zn1 and Zn2 seem to play important roles in the selection of correct transcription start sites (8,15,43).…”

Yeast RNA Polymerase II Lacking the Rpb9 Subunit Is Impaired for Interaction with Transcription Factor IIF

“…These results suggested that the underlying biochemical basis for the alterations in the positions of mRNA 5Ј-ends conferred by the ⌬Rpb9 RNAPII involves an altered interaction involving the mutant polymerase and one or more of the purified general transcription factors in the reconstituted reactions. In this regard, it was recently reported that a GAL4-Rpb9 fusion protein, which contained the GAL4 DNA binding domain fused to a truncated Rpb9 lacking the N-terminal 32 amino acids, interacted with the large subunit of yeast TFIIE (Tfa1) in a yeast two-hybrid assay (28). Although this result suggests that Rpb9 might physically interact with TFIIE, the detected interaction in the two-hybrid assay was reported to be very weak with a GAL4-Rpb9 fusion protein containing the entire Rpb9 coding sequence.…”